The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules. Such molecules are useful in biotechnology, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines.
With the invention in mind, a search of the prior art was conducted. E. coli plasmids have long been the single most important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to the patient.
Today, the FDA standards are not defined except in preliminary form (see: FDA Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications, 1996). However, in the future, international standards for plasmid DNA purity are likely to be the same or very similar to those that are used for recombinant protein products similarly produced from E. coli fermentation, and such standards exceed the current purity attainable from established methods. Most glaringly, the accepted standard of <100 pg host genomic DNA per dose (see: FDA Points to consider in the characterization of cell lines used to produce biologics, 1993) is far below the levels currently attainable for purified plasmid preparations (100 pg per 1 mg dose is equivalent to one part per ten million).
The basic methods for obtaining plasmids (by bacterial fermentation), and for their purification (e.g., by the alkaline lysis method (Bimboim, H C, Doly J. 1979, Nucleic Acids Res. 7: 1513-1523)) are well-known. Initially, the fermented bacterial cell paste is resuspended and lysed (using a combination of sodium hydroxide and sodium dodecylsulfate), after which the solution is neutralized by the addition of acidic salt (e.g., potassium acetate), which precipitates the bacterial DNA and the majority of cell debris. The bulk of super-coiled plasmid DNA remains in solution, along with contaminating bacterial RNA, DNA and proteins, as well as E. coli endotoxin (lipopolysaccharide, or LPS). The soluble fraction is then separated by filtration and subjected to a variety of purification steps, which may include: RNase digestion; chromatography (ion exchange gel filtration, hydroxyapatite, gel filtration, hydrophobic interaction, reverse phase, HPLC, etc.); diafiltration; organic extraction, selective precipitation, etc.
Clearly, increasing the purity of the starting material and achieving better downstream purification are essential goals for manufacturing clinical grade DNA on an industrial scale.
Fermentation Media Considerations
Design of a balanced medium is based on the cell's energy requirements and elemental composition.
Typically, the nutritional requirements are satisfied by either minimal media or semi-defined media.
Semi-defined media contain complex components such as yeast extract, casamino acids, and peptones. The addition of complex components supplies growth factors, amino acids, purines and pyrimidines and often supports higher cell densities.
Carbon accounts for half of the cellular composition. Accordingly, carbon is included in the highest amounts. The carbon source provides energy and biomass, and is usually utilized as the limiting nutrient. Glucose is the conventional carbon source. It is metabolized very efficiently and therefore gives a higher cellular yield. However, high glucose concentrations cause undesirable acetate production due to metabolic overflow (known as the Crabtree effect). Glycerol is also used and is often the preferred carbon source in batch cultures. Although cellular yields from glycerol are slightly smaller than from glucose, glycerol does not cause as high of levels of acetate production and can be used at higher concentrations without being inhibitory. Glycerol also supports reduced maximum specific growth rates.
The requirement for nitrogen may be satisfied by inorganic or organic nitrogen sources. Ammonia and ammonium salts (e.g. NH4Cl, (NH4)2SO4) are used in minimal media. Semi-defined media supply nitrogen either partly or entirely from complex components, including yeast extract, peptones, and casamino acids.
Minerals are required for growth, metabolism, and enzymatic reactions. Magnesium, phosphorus, potassium, and sulfur are typically added as distinct media components. Di- and monopotassium phosphates provide potassium and phosphorous and also function as buffering agents in certain proportions. Magnesium sulfate heptahydrate is often used as the source of magnesium and sulfur. Other essential minerals include calcium, copper, cobalt, iron, manganese, molybdenum and zinc. These are required in smaller amounts and are often supplied by addition of a trace minerals solution, though they are usually present as impurities in the major ingredients. Osmolarity is adjusted with sodium chloride.
The use of animal-derived products, and in particular bovine products, in plasmid manufacture is unacceptable due to the risk of prion or virus contamination. All media components should be certified animal product free. Vegetable-derived substitutes are available for many components which have animal origin (e.g. vegetable glycerol, soy peptone).
Plasmid Fermentation Process Considerations
Growth Rate
The use of reduced growth rate is the unifying principle in high quality, high yield plasmid fermentations. High growth rates have been associated with acetate production, plasmid instability, and lower percentages of super-coiled plasmid. A reduced growth rate alleviates growth rate-dependent plasmid instability by providing time for plasmid replication to synchronize with cell division.
Growth Conditions
Fermentation gives us the ability to control and monitor many of the parameters that affect plasmid quality and yield. Super-coiling is known to be affected by oxygen and temperature (Dorman C J et al. 1988 J. Bacteriol. 179: 2816-2826), (Goldstein E, Drlica K. 1984 Proc Natl Acad Sci USA. 81: 4046-4050). Oxygen has been shown to play a role in plasmid stability. One study (Hopkins D J, Betenbaugh M J, Dhurjati P. 1987 Biotechnol Bioeng. 29: 85-91) found that a single drop in dissolved oxygen concentration to 5% of air saturation led to rapid loss in plasmid stability. Another study (Namdev P K, Irwin N, Thompson B G, Gray M R. 1993 Biotechnol Bioeng. 41: 666-670) showed that fluctuations in oxygen input lead to plasmid instability. Furthermore, the formation of nicked plasmids and multimers can be affected by many parameters, including temperature, pH, dissolved oxygen, nutrient concentration, and growth rate (Durland R H, Eastman E M. 1998 Adv Drug Deliver Rev. 30: 33-48). The optimal temperature for E. coli growth is 37° C. However, lower temperatures (30-37° C.) may be used in batch fermentation to cause a reduced maximum specific growth rate. Higher temperatures (36-45° C.) can also be employed to induce selective plasmid amplification with some replication origins such as pUC, and pMM1 (Wong E M, Muesing M A, Polisky, B. 1982 Proc Natl Acad Sci USA. 79: 3570-3574), (Lin-Chao S, Chen W T, Wong T T. 1992 Mol. Microbio. 6: 3385-3393) and runaway replicon R plasmids. Hamann et. al. 2000 (Hamann C W, Nielsen J, Ingerslev E. 2000 World Patent Application WO0028048) report a process for the production of R plasmids wherein plasmid production is maintained at a low level (by use of low temperature) to avoid retardation of growth due to plasmid DNA synthesis; once the host cell population is high, plasmid production is induced by temperature shifting.
Batch Fermentation
Batch fermentation has the main advantage of simplicity. All nutrients that will be utilized for cell growth and plasmid production throughout the culture period are present at the time of inoculation. A batch fermentation has a lag phase, exponential growth phase, and stationary phase. The use of a suitable inoculum (1-5% of the culture volume) will reduce the length of the lag phase. During the exponential phase all nutrients are in excess; thus the specific growth rate will be essentially the maximum specific growth rate, μmax, as predicted by Monod kinetics. As discussed previously, reduced growth rates are desirable for plasmid production. In batch fermentation the growth rate can only be reduced by reducing μmax. This has been achieved by growth at lower temperatures and by growth on glycerol instead of glucose. Batch fermentation at 30° C. using glycerol will typically result in μmax≦0.3 h−1, which is sufficient to prevent deleterious acetate accumulation and growth rate associated plasmid instability (Thatcher D R, Hitchcock A, Hanak J A J, Varley D L. 2003 U.S. Pat. No. 6,503,738). Glycerol can also be used at much higher concentrations than glucose without being inhibitory, leading to higher biomass yields. Generally, biomass yields of up to 60 g/L DCW can be obtained with batch fermentation.
Fed-Batch Fermentation
Fed-batch fermentation is especially useful for plasmid production. Controlled addition of a limiting nutrient allows for control of growth rate at rates <μmax. Also, fed-batch fermentation results in higher yields. The key to fed-batch fermentation is supplying substrate at a rate such that it is completely consumed. As a result, residual substrate concentration is approximately zero and maximum conversion of substrate is obtained. Metabolic overflow from excess substrate is avoided, reducing the formation of inhibitory acetate.
Fed-batch fermentation starts with a batch phase. Cells are inoculated into an initial volume of medium that contains all non-limiting nutrients and an initial concentration of the limiting substrate. Controlled feeding of the limiting nutrient begins once the cells have consumed the initial amount of substrate.
One of the simplest and most effective feeding strategies is exponential feeding. This method allows the culture to grow at a predetermined rate less than μmax without the need of feedback control. The fermentation begins with a batch mode containing a non-inhibitory concentration of substrate. The cells grow at μmax until the substrate is exhausted, at which point the nutrient feeding begins.
The DO-stat and pH-stat methods are fairly easy to implement since most standard fermentor systems include dissolved oxygen and pH monitoring. Trends in dissolved oxygen (DO) and pH can indicate whether substrate is available to the cells. Exhaustion of substrate causes decreased oxygen uptake and the DO concentration in the medium rises. The pH also rises due to consumption of metabolic acids. Feeding is triggered when DO or pH rises above a set threshold. The growth rate can be adjusted by changing the DO or pH threshold value.
Exemplary Plasmid Fermentation Processes
Examination of current yields reveal that typical laboratory shake flask culture produces from 1-5 mg of plasmid DNA/L of culture, whereas a computer controlled fermentor can produce, typically, from 10-250 mg/L.
Lahijani et al. (Lahijani R, Hulley G, Soriano G, Horn N A, Marquet M. 1996 Human Gene Therapy 7: 1971-1980) have reported using a pBR322-derived plasmid with a temperature sensitive single point mutation (pUC origin) in a fermentation with exponential feeding and a temperature shift from 37° C. to 42-45° C. They achieved a plasmid yield of 220 mg/L in a 10 L fermentor. The same plasmid without the mutation in batch fermentation (pBR322 derived origin) at 30° C. yielded only 3 mg/L plasmid. Friehs et al. (Friehs K, Flaschel E, Schleef M, Schmidt T. 2003 U.S. Pat. No. 6,664,078) describe a fed-batch process using a glycerol yeast extract medium with DO-stat feedback controlled feeding. The fermentation started with an initial batch volume of 7.5 L. Agitation was increased to keep DO above 30%. Feed medium was pumped in when DO reached a threshold setpoint of 45%. The culture reached stationary phase after 41 hours, yielding 60 g/L DCW and 230 mg/L of plasmid. Chen (Chen, W. 1999 U.S. Pat. No. 5,955,323) used a fed-batch process in semi-defined medium with combination DO-stat and pH-stat feedback control. DO and pH threshold setpoints were 50% and 7.2, respectively. When DO dropped below 30% because of high metabolic activity agitation speed was increased by a percentage of the previous speed. In a 7 L fermentor, this strategy led to a specific growth rate of 0.13 h−1 and plasmid yields of 82-98 mg/L. Durland and Eastman, Supra, 1998 report batch fermentation at 37° C. in a proprietary medium. Their process typically yields 130 mg/L and has yielded as high as 250 mg/L.
Even in view of the prior art, there remains a need for a cost effective method for high purity plasmid DNA production. The fermentation media and processes described above incorporate what is currently known in the art to improve plasmid productivity, such as reduced growth rate and plasmid copy number induction with high temperature. These processes plateau at about 200-250 mg plasmid DNA/L. This low yield imposes a cost and purity burden on commercialization of plasmid DNA production processes. Although economies of scale will reduce the cost of DNA significantly in the future, a far more economical solution to this problem is needed in order to achieve the desired cost. As well, international standards for plasmid DNA purity are likely to be the same or very similar to those that are used for recombinant protein products similarly produced from E. coli fermentation, and such standards exceed the current purity attainable from established methods. Increasing the yield (mg of DNA/gram of cell paste) in fermentation would both decrease the cost and increase the purity of the DNA (because it reduces the amount of material being processed).